Expression and Purification of Botulinum Neurotoxin Type ‘A’ Toxin Domain in E. coli and its Application in Detection

Botulism is a neuroparalytic disease caused by botulinum toxins type A-G. It has been listed as category ‘A’ biowarfare agent by CDC due to its extreme toxicity.. In the present study, we developed a simple method for the pro-duction and purification of recombinant toxin domain of BoNT type ‘A’ from Escherichia coli and established its application in BoNT detection. The BoNT/A LC gene was cloned in pET28b+ vector and expressed in E.coli. The recom-binant BoNT/A LC protein expression was achieved at 25°C with the induc-tion of 0.5mM IPTG at 16 h. The recombinant protein was purified under native conditions with more than 98% purity using simple affinity chroma-tography and confirmed by Mass spectrometry and Western blot analysis. Generated polyclonal antibodies and the toxin domain elicited a strong im-mune response in mice and rabbit. Optimized the Sandwich ELISA system resulted with the sensitivity of 7.5 ng/ml for the detection of BoNT/A. The limit of detection (LOD) in different food matrices were studied through spik-ing studies using rBoNT/A LC protein as antigen and achieved a detection limit ranging from7.5 ng/ml to 250 ng/ml. The developed method has the potential for the industrial production towards its application in detection, development of candidate vaccine molecule and production of neutralizing antibodies. Current study explored for detection of BoNT/A.

Synthesis of Silver Nanostructures and their Application in Highly Sensitive SERS Sensors

<p class="p1">A comparison of Surface Enhanced Raman Scattering (SERS) activity of chemically synthesised silver nanostructures with different shapes is reported. The silver nanostructures of cubical, prism and wire like morphology were synthesised using chemical synthesis route and utilised as SERS substrates. The sensors were fabricated by spin coating these materials over a Silicon or glass substrate. The fabricated sensors were used to analyse response with two different analytes, 4-Mercaptobenzoic acid and Rhodamine 6G under different concentrations. The signal enhancement was compared with a silver coated thin film over glass substrate and it was observed that the enhancement of the order of 10<span class="s1">³</span>is achieved. The nanowire performed better than the other forms of silver and gave a higher signal enhancement for all the analytes as compared to other nanostructures. The fabricated sensors may be useful for various applications including explosive and biowarfare agent detection.</p>

Benzoxazoles, Phthalazinones, and Arylurea-Based Compounds with IMP Dehydrogenase-Independent Antibacterial Activity against Francisella tularensis

ABSTRACT Francisella tularensis is the causative agent of tularemia and a potential biowarfare agent. The virulence of F. tularensis is decreased by deletion of guaB, the gene encoding IMP dehydrogenase (IMPDH), suggesting that this enzyme is a target for antibacterial design. Here we report that F. tularensis growth is blocked by inhibitors of bacterial IMPDHs. Seventeen compounds from two different frameworks, designated the D and Q series, display antibacterial activities with MICs of <1 μM. These compounds are also active against intracellular infections. Surprisingly, antibacterial activity does not correlate with IMPDH inhibition. In addition, the presence of guanine does not affect the antibacterial activity of most compounds, nor does the deletion of guaB. These observations suggest that antibacterial activity derives from inhibition of another target(s). Moreover, D compounds display antibacterial activity only against F. tularensis, suggesting the presence of a unique target or uptake mechanism. A ΔguaB mutant resistant to compound D73 contained a missense mutation (Gly45Cys) in nuoB, which encodes a subunit of bacterial complex I. Overexpression of the nuoB mutant conferred resistance to D73 in both wild-type and ΔguaB strains. This strain was not resistant to Q compounds, suggesting that a different off-target mechanism operates for these compounds. Several Q compounds are also effective against Mycobacterium tuberculosis, in which a second target has also been implicated, in addition to IMPDH. The fortuitous presence of multiple targets with overlapping structure-activity relationships presents an intriguing opportunity for the development of robust antibiotics that may avoid the emergence of resistance.

Application of a Broad-Range Resequencing Array for Detection of Pathogens in Desert Dust Samples from Kuwait and Iraq

ABSTRACTA significant percentage of the human population is exposed to high levels of naturally occurring airborne dusts. Although the link between airborne particulate inhalation and a variety of respiratory diseases has long been established, little is known about the pathogenic role of the microbial component of the dust. In this study, we applied highly multiplexed PCR and a high-density resequencing microarray (RPM-TEI version 1.0) to screen samples of fine topsoil particles and airborne dust collected in 19 locations in Iraq and Kuwait for the presence of a broad range of human pathogens. The results indicated the presence of potential human pathogens, includingMycobacterium,Brucella,Coxiella burnetii,Clostridium perfringens, andBacillus. The presence ofCoxiella burnetii, a highly infectious potential biowarfare agent, was confirmed and detected in additional samples by use of a more sensitive technique (real-time PCR), indicating a high prevalence of this organism in the analyzed samples. The detection of potentially viable pathogens in breathable dusts from arid regions of Iraq and Kuwait underscores the importance of further study of these environments.

The use of rapid diagnostic kits for the rapid presumptive identification of bioterrorist agents

The events of 11 September 2001 and the subsequent US anthrax mail attacks placed enormous pressure on emergency response agencies to be able to rapidly assess the potential risk of an incident as a possible case of bioterrorism. In response to this perceived need to rapidly identify bioterrorist agents in the field, a number of hand-held ?tickets? appeared in the market and were promoted heavily to emergency response personnel. These included products from Tetracore (Guardian Bio-Threat Alert System), Alexis, Sigma-Aldrich (BADD ? Biowarfare Agent Detection Device) and RAMP. This brief discussion will examine the current knowledge on such devices and their applications for the detection of bioterrorist agents.

Optimized Silver Film over Nanosphere Surfaces for the Biowarfare Agent Detection Based on Surface-Enhanced Raman Spectroscopy

This work presents the rapid detection of Bacillus subtilis spores, harmless simulants for Bacillus anthracis, using surface-enhanced Raman spectroscopy (SERS) on silver film over nanosphere (AgFON) substrates. Calcium dipicolinate (CaDPA), a biomarker for bacillus spores, can be extracted effectively from spores with nitric acid and successfully detected by SERS. The highly tunable nature of AgFON optical properties was exploited to establish general optimization conditions. AgFON surfaces optimized for 750-nm laser excitation have been characterized by UV-vis diffuse reflectance spectroscopy. The SERS signal from extracted CaDPA was evaluated over the spore concentration range 10-15-10-12M to determine the adsorption capacity of the AgFON surface and the limit of detection (LOD). These sensing capabilities have been successfully transitioned to an inexpensive, portable Raman spectrometer. Using the extraction method and this field-portable instrument, the anthrax infectious dose of 104spores were detected with only a 5-second collection period on a one-month-old prefabricated AgFON substrate.

Rapid, Automated Nucleic Acid Probe Assays Using Silicon Microstructures for Nucleic Acid Concentration

A system for rapid point-of-use nucleic acid (NA) analysis based on PCR techniques is described. The extraction and concentration of DNA from test samples has been accomplished utilizing silicon fluidic microchips with high surface-area-to-volume ratios. Short (500 bp) and medium size (48,000 bp) DNA have been captured, washed, and eluted using the silicon dioxide surfaces of these chips. Chaotropic (GuHCl) salt solutions were used as binding agents. Wash and elution agents consisted of ethanol-based solutions and water, respectively. DNA quantities approaching 40 ng/cm2 of binding area were captured from input solutions in the 100–1000 ng/mL concentration range. For dilute samples of interest for pathogen detection, PCR and gel electrophoresis were used to demonstrate extraction efficiencies of about 50 percent, and concentration factors of about 10× using bacteriophage lambda DNA as the target. Rapid, multichannel PCR thermal cycling modules with integrated solid-state detection components have also been demonstrated. These results confirm the viability of utilizing these components as elements of a compact, disposable cartridge system for the detection of NA in applications such as clinical diagnostics, biowarfare agent detection, food quality control, and environmental monitoring.